Our overall goal in this project is to characterize the functional contributions of cyclin-dependent kinase 5 (Cdk5)-mediated neuronal phosphorylation to the nervous system. Neuronal phosphorylation plays a crucial role in neuronal development and function, such as sensory recognition, behavioral control, memory, and learning. Most importantly, recent evidence suggests that abnormal phosphorylation in neurons is involved in the pathogenic mechanisms underlying many neurological diseases and drug addiction. Our main focus has been directed toward elucidating the contribution of Cdk5 in brain development and function. Cdk5, a member of a large family of proline-directed serine/threonine protein kinases, was originally cloned by homology to other members of the Cdk family that are major regulators of cell-cycle progression. However, the involvement of Cdk5 in cell-cycle regulation has never been identified. Instead, it appears to play a major role in the nervous system. The neuronal specificity of Cdk5 kinase activity is achieved through the association with its activators, either p35 or p39, which are predominantly expressed in postmitotic neurons. To date, about 40 proteins with diverse functions have been identified as substrates of Cdk5, implicating its role in the regulation of a wide range of neuronal functions. Apart from the critical role of Cdk5 in normal brain functions, deregulation of Cdk5 is involved in the pathogenesis of neurodegenerative diseases such as Alzheimer?s disease, amyotrophic lateral sclerosis (ALS), Parkinson?s disease, and drug addiction. We have used a variety of functional genomic techniques and molecular approaches, including conventional and conditional gene targeting, analysis of protein-protein interactions, as well as behavioral tests in mice to address these questions. We believe our present studies will not only contribute to a greater understanding of the molecular basis of Cdk5 functions in the brain, but also will help future efforts toward the development of more effective diagnosis and treatments for debilitating neurodegenerative and addictive diseases. Cocaine increases synaptic dopamine levels in the striatum and alters gene expression in the dopaminoceptive neurons by activating intracellular pathways that propagate the initial signal from the dopamine D1 receptor to the nucleus. Chronic exposure to cocaine upregulates several transcription factors, resulting in the long-lasting changes in gene expression that are thought to underlie neuronal adaptations in cocaine addiction. Delta-FosB, identified as such a transcription factor, has been shown to enhance the behavioral responsiveness of animals to cocaine. Therefore, identification of the target genes that are regulated by delta-FosB induction is expected to contribute to a greater understanding of the molecular mechanism underlying cocaine addiction. Recently, chronic treatment of animals with cocaine has been shown to upregulate the expression of Cdk5 and its activator p35 in the striatum through the induction of delta-FosB. Inhibition of Cdk5 activity results in increased dopamine release in the striatum, indicating the presynaptic function of Cdk5 as a negative regulator of dopamine release. Furthermore, Cdk5 modulates the efficacy of postsynaptic dopamine signaling by phosphorylating DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, molecular mass 32 kDa) at Thr 75, which converts DARPP-32 into an inhibitor of cAMP-dependent kinase (PKA). These observations suggest that Cdk5 and p35 are downstream regulators of the prolonged activation of dopamine signaling following chronic exposure to cocaine, and hence in cocaine addiction. However, the direct effects of either Cdk5 or p35 induction on striatal dopamine signaling remained to be determined in vivo. To address this question, we generated two independent transgenic mouse lines in which either Cdk5 or pp35 was overexpressed specifically in neurons under the control of the p35 promoter. Our findings indicated that Cdk5 activity was upregulated with the increased levels of p35 protein, but not with the increased levels of Cdk5 protein, suggesting that the level of p35 protein is rate-limiting for the upregulation of Cdk5 activity. We provided in vivo evidence that increased Cdk5 activity as a result of p35 overexpression leads to attenuation of cocaine-mediated dopamine signaling to the nucleus accumbens through an inhibition of the PKA and extracellular signal-regulated kinase (ERK)-cascades. Over the last decade, over 40 functionally diverse proteins have been identified as Cdk5 substrates. Some of these include neurofilaments (NF-H, NF-M), MAP1b, MAP2 and tau, catenin, presenilin-1, DARPP-32, PAK1 kinase, Munc-18, synapsin and amphiphysin. Cdk5 functions include microtubule stability, cell-cell adhesion, dopamine signaling, regulation of actin dynamics through PAK1 signaling and synaptic transmission. Recent studies have also implicated Cdk5 activity in crosstalk with other kinase signal transduction pathways such as the Extracellular Signal Regulated Kinase 1/2, c-Jun N-terminal Kinase-3 and neuregulin/Akt pathways. To further understand this aspect of Cdk5 function, we sought to identify proteins from signal transduction pathways and identify their crosstalk with Cdk5. We used the yeast two-hybrid system to search for novel interacting proteins of p35 to further understand the functions of Cdk5 in the adult nervous system. RasGRF2 was identified as a novel interacting protein and substrate of p35/Cdk5. We showed that p35/Cdk5 phosphorylates RasGRF2 on Serine737, thereby modulating the Rac signaling cascade affecting ERK1/2 activity and the subsequent distribution of both RasGRF2 and MAP1b in neurons. But Cdk5 deficiency did not rescue SOD mutant neuropathy. Cdk5 in neurodevelopment and neurodegeneration have been studied extensively, but regulation of Cdk5 activity has remained largely unexplored. We have found that Cdk5 acting via NMDA or kainate receptors can induce a transient Ca(2+)/calmodulin-dependent activation of Cdk5 that results in enhanced autophosphorylation and proteasome-dependent degradation of a Cdk5 activator, p35, and thus ultimately down-regulation of Cdk5 activity. The relevance of this regulation to synaptic plasticity was examined in hippocampal slices using theta burst stimulation. p35(-/-) mice exhibited a lower threshold for induction of long-term potentiation. Thus excitatory glutamatergic neurotransmission regulates Cdk5 activity through p35 degradation, and this pathway may contribute to plasticity. In the developing brain, the organization of the neuroepithelium is maintained by a critical balance between proliferation and cell-cell adhesion of neural progenitor cells. The molecular mechanisms that underlie this are still largely unknown. Through analysis of a conditional knockout mouse for the Kap3 gene, we show that post-Golgi transport of N-cadherin by the KIF3 molecular motor complex is crucial for maintaining this balance. N-cadherin and beta-catenin associate with the KIF3 complex by co-immunoprecipitation, and colocalize with KIF3 in cells. Furthermore, in KAP3-deficient cells, the subcellular localization of N-cadherin was disrupted. Taken together, these results suggest a potential tumour-suppressing activity for this molecular motor. Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in renal physiology, including the prevention of proteinuria. Cdk5 has been shown to influence several cellular processes in other terminally differentiated cells, in particular neurons. We examined the role of Cdk5 in podocyte differentiation, proliferation, and morphology. In conditionally immortalized mouse podocytes in culture, Cdk5 increased in association with podocyte differentiation.